Phenylketonuria (PKU) (OMIM 261600, ORPHA716) is an inherited metabolic disorder with an incidence of 1:10,000 in Europe. In most cases, this is an amino acid metabolism disorder resulting from an absent or impaired function of the liver enzyme phenylalanine hydroxylase (PAH). Deficiencies in PAH in turn result in an excess of phenylalanine (Phe) in the brain and plasma. The deficiency in PAH ultimately manifests in a lack of tyrosine, which is a precursor for neurotransmitters. Together with mutations involving enzymes of the pterin metabolism, PKU is associated with hyperphenylalaninemia (HPA).
The disease is commonly diagnosed in most countries right after birth during newborn screenings due to elevated blood Phe levels. Left undetected and untreated early in life, PKU leads to irreversible damage of the infant's nervous system, severe mental retardation and poor brain development. Features other than intellectual disabilities in untreated patients include neurological complications, neuropsychological impairments as well as executive function deficits. It has been reported that when left untreated an infant suffers a loss of IQ within the first year of infancy. Depending on the age at start of treatment, the blood Phe levels during different age periods and the compliance of the dietary therapy PKU is invariably accompanied by at least some loss of IQ. Once detected, the condition is treated by providing the infant, and later the child, with a Phe-restricted diet. In adults, the protein supplements routinely taken by classic PKU patients are Phe-free with the assumption that such adults will receive sufficient quantities of Phe through the remaining diet, controlled under a strict regimen, so that the overall diet is a low Phe diet. In particular, pregnant women who suffer from the condition are recommended to comply with a rigid Phe-limited dietary regimen to avoid the risk of impairment of the development of the foetus and congenital malformation (maternal PKU syndrome).
In more recent years it has been shown that pathological symptoms which manifest from the condition of excess of Phe, collectively termed hyperphenylalaninemia (HPA), may be divided into multiple discrete disorders, which are diagnosed according to plasma Phe concentrations and responsiveness to a co-factor for PAH. At an initial level, HPAs may be divided into HPA caused as a result of a deficiency in the cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) due to enzymatic defects in the biopterin metabolism (malignant PKU) and HPA resulting from a deficiency in PAH. The latter is further subdivided resulting in at least four sub-categories depending on the plasma concentration of Phe in the absence of dietary or other therapeutic intervention (referred to herein as “unrestricted plasma Phe concentration”) and the responsiveness to the co-factor BH4.
Normal plasma Phe homeostasis is tightly controlled resulting in a plasma Phe concentration of 60 μmol/L±15 μmol/L. Classical PKU (ORPHA79254) is the most severe form of PKU and it results from null or severe mutations in PAH, which lead to unrestricted plasma Phe concentrations greater than 1200 μmol/L when left untreated. Individuals with classical (or severe) PKU must be treated with a strict dietary regimen that is based on a very low Phe diet in order to reduce their Phe concentrations to a safe range. Milder forms of PKU also have been characterized. A less severe form of PKU is one which manifests in unrestricted plasma Phe concentrations of 10-20 mg/dL (600-1200 μmol/L) and is generally termed “mild PKU” (ORPHA79253). This moderate form of PKU is managed through the use of moderate dietary restrictions, e.g., a comparatively low-protein diet without the need of a supplementation with Phe-free amino acid formulas. Mild HPA, also referred to as benign or non-PKU-HPA (ORPHA79651) is characterized by unrestricted plasma Phe concentrations of between 180-600 μmol/L. The individuals with non-PKU-HPA are not routinely treated as they are considered to have plasma Phe levels that are within the “safe” range. In dietary PKU therapy, a range below <360 μmol/L is aimed for, with a range up to 600 μmol/L considered acceptable. Finally, BH4-responsive PKU/HPA (ORPHA293284) is characterised by unrestricted plasma Phe concentrations of >360 μmol/L which can be markedly reduced or normalized after oral loading with tetrahydrobiopterin (BH4; sapropterin dihydrochloride). This mild to moderate form of PKU/HPA is caused by specific mutations in the PAH gene leading to mutant proteins with significant residual enzymatic activity. Supplementation of BH4 as part of the PKU/HPA management enables some patients to relax their Phe-restricted dietary regimen. It is to be understood that the terms “treatment of PKU” or “PKU patient” as used herein are intended to refer to the treatment of and patients with the following forms of HPA, e.g. classical PKU, mild PKU, mild HPA and BH4-responsive PKU/HPA.
At the beginning of the dietary PKU therapy in the early 1950ies, patients have been provided with the essential amino acids (except Phe) by protein hydrolysates. Therefore, a protein with relatively high levels of essential amino acids, such as casein (a protein commonly found in mammalian milk, making up 80% of the proteins in cow milk) or bovine serum albumin was hydrolysed followed by a filtration step of the peptides to remove as much Phe contamination as possible, and/or by combining free amino acids in a mixture that includes a hydrolysed protein. Today, typically balanced mixtures of free crystalline amino acids comprising essential amino acids (except Phe) are provided to the patients. Such amino acid mixtures may have a bitter taste, cause a sandy mouthfeel and may be deemed unsuitable or undesirable for certain uses. As a result, such mixtures sometimes include flavours to mask the taste of the free amino acids and/or hydrolysed protein. In some cases, compositions in which a proportion of the amino acid content is provided by polypeptides or proteins are found to have a better taste than compositions with a high proportion of total amino acids provided as free amino acids and/or certain hydrolysed proteins. The availability of such compositions has been limited, however, because nutritional formulations have traditionally been made from protein isolated from natural food products, such as whey isolated from milk, or soy protein isolated from soy. The amino acid profiles of those proteins do not necessarily meet the amino acid requirements for a mammal. In addition, commodity proteins typically consist of mixtures of proteins and/or protein hydrolysates which can vary in their protein composition, thus leading to unpredictability regarding their nutritional value. Moreover, the limited number of sources of such proteins with a high biological value has meant that only certain combinations of amino acids are available on a large scale for ingestion in protein form.
The glycomacropeptide (GMP), a natural whey protein produced during cheese making, has been used in the treatment of PKU. GMP in its pure form lacks the aromatic amino acids phenylalanine (Phe; F), tyrosine (Tyr; Y) and tryptophan (Trp; W) as well as arginine (Arg; R), histidine (His; H) and cysteine (Cys; C) but is enriched in the large neutral amino acids isoleucine (Ile; I) and threonine (Thr; T). As a commercially available dietary protein it contains minimal amounts of Phe. However, used as single protein source in medical foods for the dietary management of PKU it has to be supplemented with Trp, Arg, Leu, His and Tyr to meet the needs of daily-required intake of these essential and semi-essential amino acids and to provide an adequate low Phe/Tyr ratio (<1). Becoming essential in PKU patients, Tyr improves their emotional behaviour dependent on the availability for the synthesis of neurotransmitters.
The present invention addresses the above issues by providing a dietary protein comprising all essential amino acids (expect Phe) that has improved properties, such as a high biological value or neutral taste. Moreover, the dietary protein may be provided as a nutritive product which can form a part of the patients' normal diet, such as baked goods, cereals or pressed bars. Alternatively, the dietary protein may be provided in a form that is suitable for the production of a nutritive product by the patient, such as pre-prepared baking mixtures or vegetable soup mixtures.
The present invention therefore aims at improving the quality of life of PKU patients, since all PKU patients must adhere to a special diet low in Phe for optimal brain development. “Diet for life” has become the standard recommended by most experts. The diet requires severely restricting or eliminating foods high in Phe, such as meat, chicken, fish, eggs, nuts, cheese, legumes, milk and other dairy products. Starchy foods, such as potatoes, bread, pasta and corn, must be monitored. The sweetener aspartame, present in many diet foods and soft drinks, must also be avoided, as aspartame consists of two amino acids: phenylalanine and aspartic acid.
Infants may still be breastfed to provide all of the benefits of breastmilk, but the quantity must also be monitored and supplementation for missing nutrients will be required. Supplementary infant formulas are used in these patients to provide the amino acids and other necessary nutrients that would otherwise be lacking in a low-phenylalanine diet. As the child grows up these can be replaced with tablets, formulas and specially formulated foods. Since Phe is necessary for the synthesis of many proteins, it is required for appropriate growth, but levels must be strictly controlled in PKU patients. In addition, tyrosine, which is normally derived from phenylalanine, must be supplemented in the diet of PKU patients.
The oral administration of tetrahydrobiopterin (or BH4) (a co-factor for the oxidation of phenylalanine) can reduce blood levels of Phe in certain patients. A tablet preparation of the compound sapropterin dihydrochloride (Kuvan®), which is a form of tetrahydrobiopterin, is commercially available. Kuvan® is the first drug that can help BH4-responsive PKU patients (ORPHA293284, depending on the clinical setting defined among clinicians as about 25-50% of the PKU population) lower Phe levels to recommended ranges. Working closely with a dietitian, some PKU patients who respond to Kuvan® may be able to increase the amount of natural protein they can eat. However, patients will still require a Phe-restricted diet.
In theory, synthetic polypeptide sequences comprising a desired mixture of amino acids could be designed and produced in a laboratory setting. This approach may raise various concerns, however, and is therefore not always applicable. First, skilled artisans are aware that obtaining high levels of production of such synthetic sequences may be very challenging. Second, even if such a synthetic protein were synthesized, its suitability for use in a nutritive product would be uncertain. For example, such a non-naturally occurring polypeptide could be an allergen or a toxin. Thus, natural proteins are preferred.
The replacement of Phe residues in natural proteins followed by recombinant production of those proteins has also been proposed in U.S. Pat. No. 6,495,344, relating to ovalbumin and casein, two highly abundant proteins in eggs and milk, respectively, and U.S. Pat. No. 6,004,930, which discloses gamma zeins, a class of proteins present in maize. However, replacing Phe in natural proteins is not always possible and may change the protein structure such that the protein is no longer expressed.
WO 2013/148332 relates to naturally occurring nutritive polypeptide sequences composed of combinations of amino acids that contain no Phe or low Phe, some of which are secreted. WO 2014/081884 relates to formulations of such isolated nutritive polypeptides, for example for nutritional purposes. WO 2016/046234 relates to a method for preparing a recombinant Phe-free or Phe-low protein.